Targeting the ATF6-Mediated ER Stress Response and Autophagy Blocks Integrin-Driven Prostate Cancer Progression.

Prostate cancer progression to the lethal metastatic castration-resistant phenotype (mCRPC) is driven by αv integrins and is associated with Golgi disorganization and activation of the ATF6 branch of unfolded protein response (UPR). Overexpression of integrins requires N-acetylglucosaminyltransferase-V (MGAT5)-mediated glycosylation and subsequent cluster formation with Galectin-3 (Gal-3). However, the mechanism underlying this altered glycosylation is missing. For the first time, using HALO analysis of IHC, we found a strong association of integrin αv and Gal-3 at the plasma membrane (PM) in primary prostate cancer and mCRPC samples. We discovered that MGAT5 activation is caused by Golgi fragmentation and mislocalization of its competitor, N-acetylglucosaminyltransferase-III, MGAT3, from Golgi to the endoplasmic reticulum (ER). This was validated in an ethanol-induced model of ER stress, where alcohol treatment in androgen-refractory PC-3 and DU145 cells or alcohol consumption in patient with prostate cancer samples aggravates Golgi scattering, activates MGAT5, and enhances integrin expression at PM. This explains known link between alcohol consumption and prostate cancer mortality. ATF6 depletion significantly blocks UPR and reduces the number of Golgi fragments in both PC-3 and DU145 cells. Inhibition of autophagy by hydroxychloroquine (HCQ) restores compact Golgi, rescues MGAT3 intra-Golgi localization, blocks glycan modification via MGAT5, and abrogates delivery of Gal-3 to the cell surface. Importantly, the loss of Gal-3 leads to reduced integrins at PM and their accelerated internalization. ATF6 depletion and HCQ treatment synergistically decrease integrin αv and Gal-3 expression and temper orthotopic tumor growth and metastasis. IMPLICATIONS: Combined ablation of ATF6 and autophagy can serve as new mCRPC therapeutic.

Ovarian torsion during ovarian stimulation prior to trigger followed by successful oocyte retrieval: A case report.

Objective: To report a case of ovarian torsion during ovarian stimulation prior to trigger followed by laparoscopic detorsion, trigger, and subsequent successful oocyte retrieval, as well as to review outcomes from the cycle. Case Presentation: A 32-year-old woman with a history of recurrent ovarian torsion presented with ovarian torsion during ovarian stimulation for in vitro fertilization prior to trigger injection. She underwent laparoscopic ovarian detorsion followed by trigger and oocyte retrieval.The rate of fertilization and blastocyst conversion of oocytes retrieved from the ovary affected by torsion was lower than from the contralateral ovary. Viable oocytes and an embryo were produced by the affected ovary. Conclusion: Ovarian torsion during ovarian hyperstimulation for in vitro fertilization is a rare occurrence, particularly prior to trigger. Clinicians can consider proceeding with trigger and oocyte retrieval after treating ovarian torsion in appropriately counseled patients.

Ten-Year Follow-up of Internal Neurofibroma Growth Behavior in Adult Patients With Neurofibromatosis Type 1 Using Whole-Body MRI.

BACKGROUND AND OBJECTIVES: Internal neurofibromas, including plexiform neurofibromas (PNF), can cause significant morbidity in patients with neurofibromatosis type 1 (NF1). PNF growth is most pronounced in children and young adults, with more rapid growth thought to occur in a subset of PNF termed distinct nodular lesions (DNL). Growth behavior of internal neurofibromas and DNL in older adults is not well documented; yet knowledge thereof is important for patient risk stratification and clinical trial design. The primary objective of this study was to evaluate the long-term growth behavior of internal neurofibromas in adults with NF1. Secondary objectives were to correlate tumor growth behavior with patient-specific, tumor-specific, and patient-reported variables. METHODS: In this prospective cohort study, internal neurofibromas were identified on coronal short TI inversion recovery sequences on baseline and follow-up whole-body MRIs (WBMRIs). Tumor growth and shrinkage were defined as a volume change ≥20%. The association between tumor growth and patient-specific (baseline age, sex, and genotype), tumor-specific (morphology, location, DNL presence on baseline WBMRI, and maximum standardized uptake value on baseline PET imaging), and patient-reported variables (endogenous and exogenous hormone exposure, pain intensity, and quality of life) was assessed using the Spearman correlation coefficient and Kruskal-Wallis test. RESULTS: Of 106 patients with a baseline WBMRI obtained as part of a previous research study, 44 had a follow-up WBMRI. Three additional patients with WBMRIs acquired for clinical care were included, generating 47 adults for this study. The median age during baseline WBMRI was 42 years (range 18-70). The median time between WBMRIs was 10.4 years. Among 324 internal neurofibromas, 62.8% (56% of PNF and 62.1% of DNL) shrank spontaneously without treatment and 17.1% (17.9% of PNF and 13.8% of DNL) grew. Growth patterns were heterogeneous within participants. Patient-specific, tumor-specific, and patient-reported variables (including endogenous and exogenous hormone exposure) were not strong predictors of tumor growth. DISCUSSION: Internal neurofibroma growth behavior in older adults differs fundamentally from that in children and young adults, with most tumors, including DNL, demonstrating spontaneous shrinkage. Better growth models are needed to understand factors that influence tumor growth. These results will inform clinical trial design for internal neurofibromas.

A single-cell atlas of the cycling murine ovary.

The estrous cycle is regulated by rhythmic endocrine interactions of the nervous and reproductive systems, which coordinate the hormonal and ovulatory functions of the ovary. Folliculogenesis and follicle progression require the orchestrated response of a variety of cell types to allow the maturation of the follicle and its sequela, ovulation, corpus luteum formation, and ovulatory wound repair. Little is known about the cell state dynamics of the ovary during the estrous cycle and the paracrine factors that help coordinate this process. Herein, we used single-cell RNA sequencing to evaluate the transcriptome of >34,000 cells of the adult mouse ovary and describe the transcriptional changes that occur across the normal estrous cycle and other reproductive states to build a comprehensive dynamic atlas of murine ovarian cell types and states.

Alcohol-Induced Liver Injury: Down-regulation and Redistribution of Rab3D Results in Atypical Protein Trafficking.

Previous work from our laboratories has identified multiple defects in endocytosis, protein trafficking, and secretion, along with altered Golgi function after alcohol administration. Manifestation of alcohol-associated liver disease (ALD) is associated with an aberrant function of several hepatic proteins, including asialoglycoprotein receptor (ASGP-R), their atypical distribution at the plasma membrane (PM), and secretion of their abnormally glycosylated forms into the bloodstream, but trafficking mechanism is unknown. Here we report that a small GTPase, Rab3D, known to be involved in exocytosis, secretion, and vesicle trafficking, shows ethanol (EtOH)-impaired function, which plays an important role in Golgi disorganization. We used multiple approaches and cellular/animal models of ALD, along with Rab3D knockout (KO) mice and human tissue from patients with ALD. We found that Rab3D resides primarily in trans- and cis-faces of Golgi; however, EtOH treatment results in Rab3D redistribution from trans-Golgi to cis-medial-Golgi. Cells lacking Rab3D demonstrate enlargement of Golgi, especially its distal compartments. We identified that Rab3D is required for coat protein I (COPI) vesiculation in Golgi, and conversely, COPI is critical for intra-Golgi distribution of Rab3D. Rab3D/COPI association was altered not only in the liver of patients with ALD but also in the donors consuming alcohol without steatosis. In Rab3D KO mice, hepatocytes experience endoplasmic reticulum (ER) stress, and EtOH administration activates apoptosis. Notably, in these cells, ASGP-R, despite incomplete glycosylation, can still reach cell surface through ER-PM junctions. This mimics the effects seen with EtOH-induced liver injury. Conclusion: We revealed that down-regulation of Rab3D contributes significantly to EtOH-induced Golgi disorganization, and abnormally glycosylated ASGP-R is excreted through ER-PM connections, bypassing canonical (ER→Golgi→PM) anterograde transportation. This suggests that ER-PM sites may be a therapeutic target for ALD.

The non-canonical mechanism of ER stress-mediated progression of prostate cancer.

BACKGROUND: The development of persistent endoplasmic reticulum (ER) stress is one of the cornerstones of prostate carcinogenesis; however, the mechanism is missing. Also, alcohol is a physiological ER stress inducer, and the link between alcoholism and progression of prostate cancer (PCa) is well documented but not well characterized. According to the canonical model, the mediator of ER stress, ATF6, is cleaved sequentially in the Golgi by S1P and S2P proteases; thereafter, the genes responsible for unfolded protein response (UPR) undergo transactivation. METHODS: Cell lines used were non-malignant prostate epithelial RWPE-1 cells, androgen-responsive LNCaP, and 22RV1 cells, as well as androgen-refractory PC-3 cells. We also utilized PCa tissue sections from patients with different Gleason scores and alcohol consumption backgrounds. Several sophisticated approaches were employed, including Structured illumination superresolution microscopy, Proximity ligation assay, Atomic force microscopy, and Nuclear magnetic resonance spectroscopy. RESULTS: Herein, we identified the trans-Golgi matrix dimeric protein GCC185 as a Golgi retention partner for both S1P and S2P, and in cells lacking GCC185, these enzymes lose intra-Golgi situation. Progression of prostate cancer (PCa) is associated with overproduction of S1P and S2P but monomerization of GCC185 and its downregulation. Utilizing different ER stress models, including ethanol administration, we found that PCa cells employ an elegant mechanism that auto-activates ER stress by fragmentation of Golgi, translocation of S1P and S2P from Golgi to ER, followed by intra-ER cleavage of ATF6, accelerated UPR, and cell proliferation. The segregation of S1P and S2P from Golgi and activation of ATF6 are positively correlated with androgen receptor signaling, different disease stages, and alcohol consumption. Finally, depletion of ATF6 significantly retarded the growth of xenograft prostate tumors and blocks production of pro-metastatic metabolites. CONCLUSIONS: We found that progression of PCa associates with translocation of S1P and S2P proteases to the ER and subsequent ATF6 cleavage. This obviates the need for ATF6 transport to the Golgi and enhances UPR and cell proliferation. Thus, we provide the novel mechanistic model of ATF6 activation and ER stress implication in the progression of PCa, suggesting ATF6 is a novel promising target for prostate cancer therapy.

Single-cell sequencing reveals suppressive transcriptional programs regulated by MIS/AMH in neonatal ovaries.

Müllerian inhibiting substance (MIS/AMH), produced by granulosa cells of growing follicles, is an important regulator of folliculogenesis and follicle development. Treatment with exogenous MIS in mice suppresses follicle development and prevents ovulation. To investigate the mechanisms by which MIS inhibits follicle development, we performed single-cell RNA sequencing of whole neonatal ovaries treated with MIS at birth and analyzed at postnatal day 6, coinciding with the first wave of follicle growth. We identified distinct transcriptional signatures associated with MIS responses in the ovarian cell types. MIS treatment inhibited proliferation in granulosa, surface epithelial, and stromal cell types of the ovary and elicited a unique signature of quiescence in granulosa cells. In addition to decreasing the number of growing preantral follicles, we found that MIS treatment uncoupled the maturation of germ cells and granulosa cells. In conclusion, MIS suppressed neonatal follicle development by inhibiting proliferation, imposing a quiescent cell state, and preventing granulosa cell differentiation.

Patient-reported family distress among long-term cancer survivors.

Two quality of life studies at the University of Nebraska Medical Center and 3 similar studies in the nursing literature were compared regarding family distress to illness scores as reported by long-term cancer survivors. All studies were cross-sectional mail surveys and used City of Hope National Medical Center questionnaires. Participants represented a broad range of survivorship in terms of diagnosis and length of survival (range of means 3Y8 years). Single-item scores were compared among participants regarding the item "How distressing has your illness been for your family?" Significant levels of patient-reported family distress to illness were reported in all 5 studies. Patient survivors may have been able to recall past levels of significant family distress despite prolonged survival or they may have reported significant ongoing family distress as a result of their disease and treatment. Longitudinal assessment of patients' and families' quality of life is essential throughout survivorship. Future studies should identify and compare the types distress experienced by patient survivors and families over time and also measure the intensity of their distress. Interventions designed to meet their individual and collective needs, thereby decreasing their distress, are needed to improve quality of life for survivors and families.